__________________________________________________Principal Investigator: COVIELLO, ANDREA ABSTRACT Women with polycystic ovary syndrome, a known disorder of androgen excess, are at increased risk for metabolic syndrome, type 2 diabetes, dyslipidemia and hypertension raising concern for increased cardiovascular disease risk as well. However, the relation of circulating testosterone levels with women's health outcomes in the general population is poorly understood and the subject of this investigation. Preliminary data from the women in the Offspring Cohort of the Framingham Heart Study (FHS) revealed a strong positive association between higher free testosterone and metabolic syndrome, type 2 diabetes, dyslipidemia, and high blood pressure. However, studies of the association of testosterone levels with outcomes in women have been hampered by the inaccuracy of the platform-based immunoassays in the low range prevalent in women. The overall objective of this proposal is to characterize circulating testosterone levels in women across a broad age range from a community-based population using state of the art liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The Framingham Heart Study (FHS) cohorts are ideally suited for this project given the study's community-based sample population, stored serum, and extensive clinical phenotyping. Furthermore, LC-MS/MS method provides accurate and precise measurement of testosterone concentrations in the low range found in women. We will assess the association between circulating testosterone levels and health outcomes in ~4500 women, focusing on metabolic and cardiovascular disease, in cross-sectional and longitudinal analyses over a 5-10 year period. We will first formulate a population-based reference range for total and free testosterone in women with the Framingham Heart Study cohorts measuring total testosterone by LC-MS/MS and calculating free testosterone by using updated law of mass action equations (specific aim 1). We will determine the cross-sectional clinical correlates of testosterone, including relations to prevalent metabolic dysfunction (type 2 diabetes, dyslipidemia / atherogenic lipid profiles, hypertension, metabolic syndrome, and cardiovascular disease) in women across a broad age range (specific aim 2). We will evaluate the relations of circulating total testosterone, free testosterone, sex-hormone binding globulin (SHBG) and incident metabolic (type 2 diabetes mellitus, dyslipidemia / atherogenic lipid profiles, metabolic syndrome), hypertension, and cardiovascular disease in women longitudinally (specific aim 3). We will generate reference limits for total and free testosterone levels in women based on the results of the longitudinal analysis. The proposed investigation, by furthering our knowledge of the health consequences of high testosterone levels in women, a potentially modifiable hormonal factor, will facilitate the identification of high risk individuals as well as the development of effective risk reduction strategies for metabolic dysfunction and cardiovascular disease for women across a broad age range.